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Chapter 2 - Microscopy CHAPTER 2 - MICROSCOPY Cells are studied by a combination of methods One of the most important tools used to study cell structures has been the microscope. In fact, cells were not described until 1665, when Robert Hooke examined a piece of cork using a microscope he had made. In his book Micrographia, published in 1665, Hooke drew what he saw and described many objects that he viewed through his microscope. Hooke did not actually see cells in the cork; he saw the walls of dead cork cells. Not until much later was it realized that the interior enclosed by the walls is the important part of living cells. 2.1. Light microscopes A few years after Hooke described dead cork cells, the Dutch naturalist Anto van Leeuwenhoek viewed living cells with small lenses that he made. However, he did not share his lens making techniques, and more than a century passed before biologists realized the importance of microscopes and what they could reveal. It was not until the early 19th century that microscopes were sufficiently developed for biologists to begin their study of cells. Most cells are too small to be seen. Even if you use a magnifying glass or a hand lens, a cell would only appear as big as a full stop. You need a microscope to see the detailed structure of cells. The most common type of microscope used in schools and colleges is called a light microscope. The name ‘light microscope’ means that light is passed through the specimen that is being studied. Light rays are focused on to a transparent specimen by a condenser lens. The rays pass through the specimen and are focused again by two more lenses – the objective lens and the eyepiece lens. These two lenses produce a magnified image. As many biological specimens are colourless and nearly transparent, stains are often used to make different parts show up clearly. Stains usually colour just a particular part of a cell; iodine solution, for example, colours starch grains blueblack. Some stains, such as methylene blue or iodine solution, can be added to living cells. In other cases, the specimen is ‘fixed’ by adding a chemical such as acetic acid or alcohol, known as a fixative. These chemicals react with substance in the cell, making them insoluble and so anchoring them in position. The cells are killed when the fixative is added. Stains may be added either before or after the fixing process. So long as the specimen is thin enough to allow light to pass through, there are no limitations on what you can look at using a light microscope. Living, moving organisms such as protoctists can be watched, or you can look at a permanent stained preparation of a thin section through a piece of a human tissue. 2.2 The function of the parts of the microscope Tube – the microscope image is viewed through the tube with the aid of an eyepiece (one in a monocular microscope and two in a binocular microscope) Eyepiece – the image projected by the objective is further magnified when viewed through this lens (Fig 2.1) Arm – the central element integrating all the mechanical and optical components to form the complete microscope. It supports the body tube and is the part you can grasp to carry the microscope. 29 Chapter 2 - Microscopy Objective lenses – form a magnified image of the objective in the intermediate image plane. There are three objective lenses to choose from: low power objective, medium power objective and high power objective. Stage – supports the slide which is clamped into position by means of clips. This has a hole in it that allows light to shine up through the specimen. Coarse adjustment – moves the tube and lenses up and down to approximately the right position so that the specimen is in focus. This knob is used only with the low power objective lens. Fine adjustment – moves the tube and lenses up and down to put the specimen at the right position so that the specimen is perfectly focused. It is used to achieve fine focus with the high power and medium power objectives. Fig 2.1 Light source – a bulb which supplies light, it is situated inside the base of the microscope. Iris diaphragm - a hole under the stage that regulates the amount of light that goes through a specimen on the stage, it collects light and illuminates the specimen. Mirror – has a flat surface on one side and concave surface on the other side that is used to reflect light up through the specimen on the stage. The microscope is used to see tiny objects that are invisible to the naked eyes. It uses lenses to magnify the object to focus (display) it in greater details. A very good light microscope can magnify about 1500 times, and can show many of the important structures in animal and plant cells. The discovery and the introduction of the microscope have significantly contributed (more than any other instrument) to 30 Chapter 2 - Microscopy the development and understanding of biology as a science. There are two types of microscope named after the source of illumination: light microscope and electron microscope. The light microscope is the one that you are going to use in your biology laboratory. 2.3. Electron microscopes The principle is the same as that of a light microscope except that beams of electrons are used instead of beams of light. They are focused using electromagnets rather than glass lenses. As electrons are easily stopped by air molecules, the space inside an electron microscope must be a vacuum. As our eyes do not respond to electrons, the electrons are allowed to hit a fluorescent screen which emits visible light where the electrons hit (Fig 2.2 and 2.4). Electrons cannot penetrate materials as well as light rays can, so specimens for viewing in an electron microscope must be much thinner than those used in a light microscope. This and the fact that the specimen has to be placed in a vacuum, places great limitations on what can be viewed using an electron microscope. In particular, it is not usually possible to look at living material. As in a light microscope, specimens are stained. Heavy metal ions, such as lead or osmium, are added to the specimen and are taken up by particular parts of the cells. Atoms of these metals have large, positively charged nuclei which scatter electrons rather than letting them pass straight through. These electrons therefore do not arrive on the screen, so leaving a dark area in the image. The structures in a cell which have taken up these heavy metal stains therefore appear dark. Fig 2.2 31 Chapter 2 - Microscopy 2.4 Magnification and resolution Magnification is the number of times larger an image is than the specimen. For example, if a cell is 10µm in diameter, and a microscope produces an image of it which is 1mm (1000 µm) in diameter, then the microscope has magnified the specimen 100 times. Size of image Magnification = -----------------------Size of specimen The magnification produced by a light microscope depends on the strengths of the objective lens and the eyepiece lens. If you are using a x40 objective lens and a x10 eyepiece lens, then your specimen is being magnified 400 times. There is no limit to the amount a light microscope can magnify. By putting in stronger lenses, or more lenses, you could produce a huge image several metres across. But the image would not be at all clear, and you would not be able to see any more detail than before. This is because the resolution of a light microscope is limited. Indeed, at higher magnification, the image would become blurrier. Light microscopes can effectively magnify objects only about 1000 times Resolution is the degree of detail which can be seen (Fig 2.5). The limit of resolution of a microscope is the minimum distance by which two points can be separated and still be seen as two separate points and not one fuzzy one. Newspaper photography’s, for example, have a rather poor resolution, being made up of quite large dots which you can see with the naked eye. This limits the amount of detail which you can see. Compare this with a good quality photograph with a higher resolution (smaller dots) The limit of resolution depends on the wavelength of light. The resolution limit is about 0.45 times the wavelength. Shorter wavelengths give the best (smallest) resolution. The shortest wavelength light, which we can see in blue light, has a wavelength of about 450nm. This gives a resolution of about 0.45x 450nm, which is close to 200nm. Any objects smaller than this, or any points less than 200nm apart, will either be invisible or appear as blurs. Electron beams, however, have a much shorter wavelength than light; so much better resolutions can be resolution around 400 times better than that of light microscopes, being able to separate objects as light as 0.5 nm apart. Resolution is the capacity to distinguish fine detail in an image. This is defined as the minimum distance between two points at which they can both be seen separately rather than a single, blurred point. Resolution is the ability of an optical instrument to show two close objects as separate. For e.g, what looks to your unaided eye like a single star in the sky may be resolved as two separate stars with the help of a telescope? 32 Chapter 2 - Microscopy Fig 2.3 2.5 Comparison of advantages and disadvantages of the light and electron microscopes (Fig 2.3) LIGHT MICROSCOPE ELECTRON MICROSCOPE Advantages Disadvantages Cheap to purchase and operate Small and portable-can be used almost anywhere Unaffected by magnetic fields Preparation of material is relatively quick and simple, requiring only a little expertise Material rarely distorted by preparation Natural colour of the material can be observed Disadvantages Expensive to purchase and operate Very large and must be operated in special rooms Affected by magnetic field Preparation of material is lengthy and requires considerable expertise and sometimes complex equipment Preparation of material may distort it All images are in black and white Magnifies objects up to 2000* The depth of field is restricted Magnifies objects over 500000* It is possible to investigate a greater depth of field Advantages 33 Chapter 2 - Microscopy Fig 2.4 34 Chapter 2 - Microscopy Fig 2.5 35